This project combines functional (signal transduction) and molecular (gene expression) approaches to characterize age-induced alterations in the hippocampus and the cholinergic system innervating it. The first two specific aims of this project address PROGRAM OBJECTIVE #3. In the aged Long-Evans rat, we showed that the maximal level of muscarinic receptor- mediated PI response is depressed in learning-impaired subjects. We propose in Specific Aim 1 to investigate the integrity of G-proteins, to test the hypothesis that a decline in PLC-beta is responsible for depressed MR-PI function, and to evaluate two other neurotransmitter receptors coupled to PI turnover. In a study of the hippocampal muscarinic autoreceptor, we showed that the inhibition of acetylcholine release is mediated by both M2 and M4 subtypes. In Specific Aim 2 we will examine the effect of age and behavioral impairment on the evoked release of acetylcholine and the coupling efficiency of the autoreceptor, and immunocytochemistry to ChAT will be combined with in situ hybridization histochemistry (ISHH) for m2 and m4 mRNA to confirm that multiple muscarinic receptors are expressed as autoreceptors. Using competitive RT/PCR to quantitate BAPP-695 mRNA, we showed that this marker was selectively increased in the hippocampus of the learning-impaired rat. ISHH analysis of aged rats with quantified learning impairment confirmed the elevation of BAPP mRNA and further revealed elevation of mRNAs for Mn- SOD, BDNF, b-NOS, and GFAP in the hippocampus. The striatum of the aged rat exhibited increased in mRNA similar to the hippocampus, but the pattern of effects on protein levels, assessed with immunoblotting techniques, was different form hippocampus. The ISHH and protein data suggest that the hippocampus is affected by aging differently from other brain regions and that this is correlated with behavioral deficits. We propose in Specific Aims 3 and 4 to further evaluate biochemical indicators of oxidative stress and gliosis, and to test two hypotheses of age-related glial activation (PROGRAM OBJECTIVES #2 AND #4). ISHH and immunoblotting will used to evaluate the synaptic marker synaptophysin, to determine if loss of synapses elicit astrogliosis. Alternatively, elevated serum glucocorticoids and defective hippocampal inhibitory feedback could cause astrocytosis in the aging brain. ISHH combined with immunostaining of glia will be used to ascertain the status of hippocampal glucocorticoid receptors.